Intracellular [Na<Superscript>+</Superscript>] modulates synergy between Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger and L-type Ca<Superscript>2+</Superscript> current in cardiac excitation–contraction coupling during action potentials

Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca²⁺ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX) to trigger SR Ca²⁺ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca²⁺ release, independent of effects on SR Ca²⁺ load, Ca²⁺ release was determined by recording Ca²⁺ spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca²⁺]_i fixed at 150 nmol/L. In response to AP clamps, normalized Ca²⁺ spike amplitudes (ΔF/F ₀) increased sigmoidally and doubled as [Na⁺]_i was elevated from 0 to 20 mmol/L with an EC₅₀ of ~10 mmol/L. This [Na⁺]_i-dependence was independent of I _Na as well as SR Ca²⁺ load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F ₀ amplitude in myocytes with 20 mmol/L [Na⁺]_i, but not with 5 mmol/L [Na⁺]_i. SR Ca²⁺ release was complete before the membrane repolarized to −15 mV, indicating Ca²⁺ entry into the dyad (not reduced extrusion) underlies [Na⁺]_i-dependent enhancement of ECC. Because I _Ca,L inhibition with 50 mmol/L Cd²⁺ abolished Ca²⁺ spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca²⁺ release by synergistically increasing the efficiency of I _Ca,L-mediated ECC.     

Meloxicam-loaded nanocapsules as an alternative to improve memory decline in an Alzheimer’s disease model in mice: involvement of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase

The objective of this study was to investigate the effect of meloxicam-loaded nanocapsules (M-NC) on the treatment of the memory impairment induced by amyloid β-peptide (aβ) in mice. The involvement of Na⁺, K⁺-ATPase and cyclooxygenase-2 (COX-2) activities in the hippocampus and cerebral cortex was also evaluated. Mice received aβ (3 nmol/ 3 μl/ per site, intracerebroventricular) or vehicle (3 μl/ per site, i.c.v.). The next day, the animals were treated with blank nanocapsules (17 mL/kg) or M-NC (5 mg/kg) or free meloxicam (M-F) (5 mg/kg). Treatments were performed every other day, until the twelfth day. Animals were submitted to the behavioral tasks (open-field, object recognition, Y-maze and step-down inhibitory avoidance tasks) from the twelfth day. Na⁺, K⁺-ATPase and COX-2 activities were performed in hippocampus and cerebral cortex. aβ caused a memory deficit, an inhibition of the hippocampal Na⁺, K⁺-ATPase activity and an increase in the hippocampal COX-2 activity. M-NC were effective against all behavioral and biochemical alterations, while M-F restored only the COX-2 activity. In conclusion, M-NC were able to reverse the memory impairment induced by aβ, and Na⁺, K⁺-ATPase is involved in the effect of M-NC.     

Dietary supplementation with <Emphasis Type="Italic">N</Emphasis>-acetylcysteine, α-tocopherol and α-lipoic acid prevents age related decline in Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and associated peroxidative damage in rat brain synaptosomes

This study has shown that in aged rat brain (22–24 months) crude synaptosomes in comparison to that in young animals (4–6 months), a striking decrease in the activity of Na⁺,K⁺-ATPase occurs along with decreased K _m and V _max but without any change in enzyme content as seen by immunoblotting. This is associated with an accumulation of peroxidative damage products in aged brain. When rats are given antioxidant supplementation in the diet with a combination of N-acetylcysteine, α-tocopherol and α-lipoic acid daily from 18 months onwards and sacrificed at 22–24 months for experimentation, the age associated decrease of Na⁺,K⁺-ATPase activity, alterations of its kinetic parameters and accumulation of peroxidative damage products in brain synaptosomes are prevented nearly completely. Because of the critical importance of Na⁺,K⁺-ATPase in neuronal functions, the results of this study may be of potential implications in controlling age-related functional deficits of the brain.     

Synaptic Plasma Membrane Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity is Significantly Reduced by the α-Keto Acids Accumulating in Maple Syrup Urine Disease in Rat Cerebral Cortex

The objective of the present study was to investigate the in vitro effects of the branched-chain α-keto acids accumulating in maple syrup urine disease, namely L-2-ketoisocaproic acid, L-2-keto-3-methylvaleric acid and L-2-ketoisovaleric acid on Na⁺, K⁺-ATPase activity in synaptic plasma membranes from cerebral cortex of 35-day-old rats. All keto acids significantly inhibited Na⁺, K⁺-ATPase activity at concentrations similar (1 mM) or even lower (0.5 mM) than those found in blood and cerebrospinal fluid of maple syrup urine disease patients. We also tested the effects of alanine on this enzyme activity. Alanine per se did not alter Na⁺, K⁺-ATPase activity, but totally prevented the branched-chain α-keto acids-induced Na⁺, K⁺-ATPase inhibition, indicating that alanine and the keto acids may possibly bind to the same site on the enzyme. We also observed that the branched-chain amino acids leucine, isoleucine and valine also inhibited Na⁺ K⁺-ATPase activity to a similar degree as that of the branched-chain α-keto acids and that alanine was able to fully prevent these effects. Considering that Na⁺, K⁺-ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings may be involved in the pathophysiology of the neurological dysfunction of maple syrup urine disease.     

Diastolic dysfunction and arrhythmias caused by overexpression of CaMKIIδ<Subscript>C</Subscript> can be reversed by inhibition of late Na<Superscript>+</Superscript> current

Transgenic (TG) Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) δ_C mice develop systolic heart failure (HF). CaMKII regulates intracellular Ca²⁺ handling proteins as well as sarcolemmal Na⁺ channels. We hypothesized that CaMKII also contributes to diastolic dysfunction and arrhythmias via augmentation of the late Na⁺ current (late I _Na) in early HF (8-week-old TG mice). Echocardiography revealed severe diastolic dysfunction in addition to decreased systolic ejection fraction. Premature arrhythmogenic contractions (PACs) in isolated isometrically twitching papillary muscles only occurred in TG preparations (5 vs. 0, P < 0.05) which could be completely terminated when treated with the late I _Na inhibitor ranolazine (Ran, 5 μmol/L). Force–frequency relationships revealed significantly reduced twitch force amplitudes in TG papillary muscles. Most importantly, diastolic tension increased with raising frequencies to a greater extent in TG papillary muscles compared to WT specimen (at 10 Hz: 3.7 ± 0.4 vs. 2.5 ± 0.3 mN/mm²; P < 0.05). Addition of Ran improved diastolic dysfunction to 2.1 ± 0.2 mN/mm² (at 10 Hz; P < 0.05) without negative inotropic effects. Mechanistically, the late I _Na was markedly elevated in myocytes isolated from TG mice and could be completely reversed by Ran. In conclusion, our results show for the first time that TG CaMKIIδ_C overexpression induces diastolic dysfunction and arrhythmogenic triggers possibly via an enhanced late I _Na. Inhibition of elevated late I _Na had beneficial effects on arrhythmias as well as diastolic function in papillary muscles from CaMKIIδ_C TG mice. Thus, late I _Na inhibition appears to be a promising option for diastolic dysfunction and arrhythmias in HF where CaMKII is found to be increased.     

K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca<Superscript>2+</Superscript> release and the associated diastolic contraction during β-adrenergic stimulation in rat ventricular cardiomyocytes

K201 has previously been shown to reduce diastolic contractions in vivo during β-adrenergic stimulation and elevated extracellular calcium concentration ([Ca²⁺]_o). The present study characterised the effect of K201 on electrically stimulated and spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca²⁺ release and contractile events in isolated rat cardiomyocytes during β-adrenergic stimulation and elevated [Ca²⁺]_o. Parallel experiments using confocal microscopy examined spontaneous diastolic Ca²⁺ release events at an enhanced spatiotemporal resolution. 1.0 μmol/L K201 in the presence of 150 nmol/L isoproterenol (ISO) and 4.75 mmol/L [Ca²⁺]_o significantly decreased the amplitude of diastolic contractions to ~16% of control levels. The stimulated free Ca²⁺ transient amplitude was significantly reduced, but stimulated cell shortening was not significantly altered. When intracellular buffering was taken into account, K201 led to an increase in action potential-induced SR Ca²⁺ release. Myofilament sensitivity to Ca²⁺ was not changed by K201. Confocal microscopy revealed diastolic events composed of multiple Ca²⁺ waves (2–3) originating at various points along the cardiomyocyte length during each diastolic period. 1.0 μmol/L K201 significantly reduced the (a) frequency of diastolic events and (b) initiation points/diastolic interval in the remaining diastolic events to 61% and 71% of control levels respectively. 1.0 μmol/L K201 can reduce the probability of spontaneous diastolic Ca²⁺ release and their associated contractions which may limit the propensity for the contractile dysfunction observed in vivo.     

Defective IGF2 and IGF1R protein production in embryonic pancreas precedes beta cell mass anomaly in the Goto–Kakizaki rat model of type 2 diabetes

Aims/hypothesis The Goto–Kakizaki (GK) rat is a spontaneous model of type 2 diabetes. Defective beta cell mass detectable in late fetal age precedes the onset of hyperglycaemia. Our hypothesis was that an embryonic IGF production deficiency might be involved in beta cell mass anomaly in the diabetic GK rat. To test this, we evaluated during pancreatic organogenesis: (1) the beta cell development in GK rats on embryonic day (E) 13.5 and E18.5; (2) IGF2 and IGF1 receptor (IGF1R) pancreatic protein production on E13.5 and E18.5; (3) the in vitro development of GK pancreatic rudiment on E13.5; and (4) the in vitro effect of IGF2 addition on beta cell mass. Materials and methods Beta cell quantitative analyses were determined by immunohistochemistry and morphometry. IGF2 and IGF1R pancreatic protein production was evaluated using western blot analyses. Dorsal pancreatic rudiments were dissected on E13.5, separated from surrounding mesenchyme and cultured for 7 days without or with recombinant IGF2. Results While beta cell mass was already decreased on E18.5, the differentiation of the first beta cells was in fact normal in E13.5 GK pancreas. Moreover, defective IGF2 and IGF1R protein production was detected in GK pancreatic rudiment as early as E13.5. The isolated GK pancreatic rudiment as maintained in vitro mimics the GK beta cell deficiency observed in vivo. This last approach enabled us to show that GK beta cells were fully responsive to IGF2 as far as their net growth is concerned. Conclusions/interpretation In diabetic GK rat, defective IGF2 and IGF1R protein production in embryonic pancreas precedes beta cell mass anomaly. IGF2 supplementation expands the pool of beta cells.     

Video monitoring of neovessel occlusion induced by photodynamic therapy with verteporfin (Visudyne<Superscript>®</Superscript>), in the CAM model

The aim of the present study was to monitor photodynamic angioocclusion with verteporfin in capillaries. Details of this process were recorded under a microscope in real-time using a high-sensitivity video camera. A procedure was developed based on intravenous (i.v.) injection of a light-activated drug, Visudyne((R)), into the chorioallantoic membrane (CAM) of a 12-day-old chicken embryo. The effect of light activation was probed after 24 h by i.v. injection of a fluorescent dye (FITC dextran), and analysis of its fluorescence distribution. The angioocclusive effect was graded based on the size of the occluded vessels, and these results were compared with clinical observations. The time-resolved thrombus formation taking place in a fraction of the field of view was video recorded using a Peltier-cooled CCD camera. This vessel occlusion in the CAM model was reproducible and, in many ways, similar to that observed in the clinical use of verteporfin. The real-time video recording permitted the monitoring of platelet aggregation and revealed size-selective vascular closure as well as some degree of vasoconstriction. Platelets accumulated at intravascular junctions within seconds after verteporfin light activation, and capillaries were found to be closed 15 min later at the applied conditions. Larger-diameter vessels remained patent. Repetition of these data with a much more sensitive camera revealed occlusion of the treated area after 5 min with doses of verteporfin and light similar to those used clinically. Consequently, newly developed light-activated drugs can now be studied under clinically relevant conditions.     

The insulin–melatonin antagonism: studies in the LEW.1AR1-<Emphasis Type="Italic">iddm</Emphasis> rat (an animal model of human type 1 diabetes mellitus)

Aims/hypothesis  

It is well documented that melatonin influences insulin secretion mediated by G-protein-coupled melatonin receptor isoforms MT1 and MT2, which are present in rat and human pancreatic islets, as well as in rat insulinoma cells. Recent investigations have proven that hyperinsulinaemic Goto–Kakizaki (GK) rats, which are a rat model of type 2 diabetic rats, and humans have decreased melatonin plasma levels, whereas a streptozotocin-induced rat model of diabetes developed reduced insulin levels combined with increased melatonin levels.